Summary This proposal identifies a new function for Adenomatous polyposis coli (APC), a tumor suppressor and negative regulator of Wnt signaling. Homozygous loss of APC causes polyposis and colon cancer in humans and deletion in early embryos causes multiple developmental phenotypes in model organisms such as Drosophila, zebrafish, and mouse. Apc mutations cause accumulation of -catenin, which activates Wnt target genes such as c-myc and cyclin D1. However, nuclear -catenin is not detectable in early adenomas of patients with germline APC mutations (familial adenomatous polyposis (FAP)) nor in the developing intestines of apc mutant zebrafish, suggesting that additional effectors are required downstream of APC. We find that APC directly enhances the activity of glycogen synthase kinase-3 (GSK-3), providing an unexpected mechanism for APC-dependent suppression of multiple downstream targets, including the nutrient sensor mTOR (mechanistic Target of Rapamycin) and -catenin. APC mutations reduce GSK-3 activity, thereby activating mTORC1 in diverse in vivo settings including adenomas from patients with FAP, intestinal adenomas in mice, and apc mutant zebrafish embryos. Our overall hypothesis is that APC directly activates GSK-3 and that this signaling motif regulates multiple pathways, including -catenin and mTOR signaling. This model reveals a critical, new function for APC, provides a mechanism for mTOR activation caused by APC mutations, and suggests a link between APC and other signaling pathways that are independent of Wnt/-catenin. In aim 1 of this proposal, we will map the GSK-3 activation domain of APC. In aim 2, we will explore the mechanism of mTOR regulation by APC in vivo, including FAP patients. Aim 3 explores the surprising observation that apc mutation causes a dramatic neoplastic phenotype in zebrafish within 3 days that requires mTOR activation. We will use molecular and genetic approaches in zebrafish to characterize these tumors as a highly accessible, in vivo model to explore APC signaling and tumorigenesis.